Method for reducing radio-resistance of prostate cancer cells and/or treating prostate cancer

ABSTRACT

A method for reducing the radio-resistance of prostate cancer cells in a subject in need thereof, comprising administering to the subject an effective amount of an active component, wherein the active component is a nanoparticle of a carrier encapsulated with cytolethal distending toxin subunit B (CdtB).

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Taiwanese Patent Application No.103102246, filed on Jan. 22, 2014, in the Taiwan Intellectual PropertyOffice, the disclosure of which is incorporated herein in its entiretyby reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the treatment of prostate cancer, andin particular, relates to a method for treating prostate cancer orreducing the radio-resistance of prostate cancer cells in a subject,wherein the method comprises administering to a subject in need aneffective amount of a medicament comprising an active component. Theactive component is a nanoparticle of a carrier encapsulated withcytolethal distending toxin subunit B (CdtB). The medicament could beused in combination with radiation therapy to provide an outstandingeffect on treating prostate cancer.

2. Description of the Related Art

The prostate is a male-specific genital organ. Prostate cancer hasbecome one of the most common cancers in the male population worldwide.The occurrence of prostate cancer is related to age. Along with changesin eating habits, increase in high fatty food intake and increase inaverage lifespan, the occurrence and mortality of prostate cancer hasrisen to become one of the top ten most common causes of death fromcancer. While the pathology of prostate is unclear, known etiologiesinclude genetics, diet, hormonal and environmental factors. There areusually no symptoms in the early stage of prostate cancer; however, asthe tumor invades or blocks the urinary tract or urethral neck, symptomssimilar to urinary track blocking occurs. In the advanced stage,symptoms of acute retention of urine, hematuria and aconuresis mayappear. In addition, when bone metasis occurs, patients may suffer fromsymptoms of bone pain, pathological cataclasis, anemia, and paraplegiacaused by the compression of the spinal cord.

The common methods for treating prostate cancer include surgery, hormonetherapy and radiation therapy. For patients with lymphatic metastasis orbone metastasis, prostate cancer can not be treated effectively becauseit is difficult to excise all of the metastatic cancer cells withsurgery. In addition, surgery is not always recommended for olderpatients since the postoperative recover time is slower. Becauseprostate cancer cells in the early stage are androgen-dependent,androgen stimulation is necessary for the growth and division ofprostate cancer cells. In the absence of androgen, prostate cancer cellswill regress. Therefore, hormone therapies, such as androgen suppressionor androgen ablation, are used in clinics to treat prostate cancer.However, because some prostate cancer cells will change into anandrogen-independent form after a period of time, such prostate cancercells can continuously grow despite treatment with androgen suppressionor androgen ablation therapy. Therefore, hormone therapy cannoteffectively treat prostate cancer.

In view of the above, both surgery and hormone therapy require anadditional therapeutic method, such as radiation therapy. Radiationtherapy refers to using a focused radiation beam to damage the DNAstructure of prostate cancer cells to stop the growth of cells. Anotheradvantage of radiation therapy is that it can be used to treat patientsof any age or health status. The dosage and radiating range can becomemore precise with the use of an apparatus. However, because prostatecancer cells with radio-resistance will generate and cells can growcontinuously even under radiation exposure, radiation therapy is lesseffective in the late stages of treatment.

In view of the limited therapeutic effect of current treatments forprostate cancer, it is important to provide one method or medicament toeffectively treat prostate cancer, especially to reduce theradio-resistance of prostate cancer cells and elevate the cure rate ofprostate cancer.

The inventors of the present invention found that a nanoparticle of acarrier encapsulated with cytolethal distending toxin subunit B (CdtB)can increase the radiation sensitivity of the prostate cancer cells withradio-resistance so as to reduce the radio-resistance of said prostatecancer cells. Radiation therapy can be used in combination with acarrier encapsulated with cytolethal distending toxin subunit B toprovide an increased therapeutic efficacy and reduce the recurrence ofprostate cancer in patients. Such treatment option would benefit theelderly or patients who are not suitable for surgery.

SUMMARY OF THE INVENTION

An objective of the present invention is to provide a use of an activecomponent in the manufacture of a medicament for reducing theradio-resistance of prostate cancer cells, wherein the active componentis a nanoparticle of a carrier encapsulated with cytolethal distendingtoxin subunit B (CdtB).

Another objective of the present invention is to provide a use of anactive component in the manufacture of a medicament for treatingprostate cancer, wherein the active component is a nanoparticle of acarrier encapsulated with cytolethal distending toxin subunit B and themedicament is used in combination with a radiation therapy.

Yet another objective of the present invention is to provide a methodfor reducing the radio-resistance of prostate cancer cells in a subject,comprising administering to the subject an effective amount of an activecomponent, wherein the active component is a nanoparticle of a carrierencapsulated with CdtB.

Yet another objective of the present invention is to provide a methodfor treating prostate cancer in a subject, comprising simultaneously orseparately administering to the subject radiation therapy and aneffective amount of an active component, wherein the active component isa nanoparticle of a carrier encapsulated with CdtB.

Yet another objective of the present invention is to provide apharmaceutical composition for reducing the radio-resistance of prostatecancer cells, comprising an effective amount of an active component,wherein the active component is a nanoparticle of a carrier encapsulatedwith CdtB.

Yet another objective of the present invention is to provide apharmaceutical composition for treating prostate cancer, comprising aneffective amount of an active component, wherein the pharmaceuticalcomposition is to be used in combination with a radiation therapy, andthe active component is a nanoparticle of a carrier encapsulated withCdtB.

The detailed technology and preferred embodiments implemented for thepresent invention are described in the following paragraphs accompanyingthe appended drawings for people skilled in this field to wellappreciate the features of the claimed invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a picture showing the nanoparticles of a carrier encapsulatedwith CdtB according to an embodiment of the present invention.

FIG. 1B is a picture showing the nanoparticles of a carrier withoutbeing encapsulated with CdtB.

FIG. 2 is a bar diagram showing the particle size of the nanoparticle ofa carrier encapsulated with CdtB according to an embodiment of thepresent invention, wherein the vertical axis represents the number (%)of the nanoparticles, and the horizontal axis represents the particlesize (nm) of the nanoparticles.

FIG. 3 shows the comparison of the survival rate of the radio-resistantprostate cancer cells treated differently (* represent the p valuesmaller than 0.01, with a significant difference), wherein the verticalaxis represents the cell survival rate (%) as compared to the controlgroup, and wherein the control group is the prostate cancer cellstreated with the nanoparticles of a carrier without being encapsulatedwith CdtB.

DETAILED DESCRIPTION OF THE INVENTION

The following will describe some embodiments of the present invention indetail. However, without departing from the spirit of the presentinvention, the present invention may be embodied in various embodimentsand should not be limited to the embodiments described in thespecification. In addition, unless otherwise stated herein, expressions“a”, “the”, and the like recited in this specification (especially inthe claims) should include both the singular and plural forms.Furthermore, the term “effective amount” used in this specificationrefers to the amount of the compound that can at least partiallyalleviate the condition that is being treated in a suspected subjectwhen administered to the subject. The term “subject” used in thisspecification refers to a mammalian, including human and non-humananimals.

Campylobacter jejuni is one of the major pathogenic bacteria causinghuman enterogastritis. It has been proven that cytolethal distendingtoxin (Cdt) plays a very important role in the pathogenic progress ofCampylobacter jejuni. This toxin could make cells swell and stop at theGap 2 phase/Mitosis phase (G2/M phase), and cause cell apoptosis. TheCdt is a holotoxin, which consists of three subunits, including CdtA,CdtB, and CdtC. The CdtA and CdtC serve the function of connecting tothe cell membrane. The CdtB is a subunit responsible for enzyme activityin Cdt, and has the activity of type I deoxyribinuclease (DNase I) whichcould damage DNA and pause cell cycle.

The inventors of the present invention found that the nanoparticle of acarrier encapsulated with CdtB is useful in increasing the sensitivityof radio-resistant prostate cancer cells to radiation and thus couldreduce the radio-resistance of the prostate cancer cells. A combined useof the nanoparticle of a carrier encapsulated with CdtB and radiationcan provide a significant effect on treating prostate cancer.

Therefore, the present invention relates to the applications of ananoparticle of a carrier encapsulated with CdtB on treating prostatecancer, including the use of he nanoparticle of a carrier encapsulatedwith CdtB in the manufacture of a medicament for reducing theradio-resistance of prostate cancer cells which are resistant toradiation and/or for treating prostate cancer; administering to asubject in need a nanoparticle of a carrier encapsulated with CdtB toreduce the radio-resistance of prostate cancer cells in the subjectand/or treat prostate cancer; and providing a pharmaceutical compositioncomprising a nanoparticle of a carrier encapsulated with CdtB, whereinthe pharmaceutical composition is used for reducing the radio-resistanceof the prostate cancer cells in a subject or used in combination with aradiation therapy to treat prostate cancer.

The CdtB involved in the present invention can be cloned and purifiedfrom Campylobacter jejuni. The method can be seen in Lin et al.,Cholesterol depletion reduces entry of Campylobacter jejuni cytolethaldistending toxin and attenuates intoxication of host cells, Infect.Immun. 79(9), 3563-3575 (2011), which is entirely incorporated hereinfor reference.

Over recent years, nanoparticles have been applied to various fields,such as the biomedical, optics and electronics. In many medical studies,nanoparticles have been used to encapsulate and deliver medicaments dueto its small particle size. In the present invention, the CdtB is in theform of a nanoparticle.

In the present invention, the carrier that is used to encapsulate CdtBis a biocompatible material. For example, the carrier may be one or morebiocompatible materials selected from the group consisting of chitosan,heparin, polyglutamic acid, tripolyphosphate, polyacrylic acid andpolyethylene glycol-chitosan. In some embodiments of the presentinvention, chitosan and heparin are used for encapsulating CdtB toprovide a desired nanoparticle.

The nanoparticle of a carrier encapsulated with CdtB of the presentinvention can be prepared by any suitable method. For example, in oneembodiment of the present invention, the desired nanoparticle could beprovided by mixing the CdtB and the carrier material in room temperaturethrough a simple ionic gelation method.

The size of the nanoparticle of a carrier encapsulated with CdtB of thepresent invention is usually not greater than 1000 nm, and preferablynot greater than 500 nm. Optionally, the size of the nanoparticle couldbe adjusted by changing the relative amount ratio of the carrier andCdtB. Generally, a higher relative amount of CdtB would provide a biggernanoparticle. For example, when using 0.2 ml heparin solution (2.0mg/ml) and 2 ml chitosan solution (1.5 mg/ml) as the carrier constituentand using 0.2 ml CdtB with the concentration of 4.0 mg/ml, 8.0 mg/ml, or16.0 mg/ml, the average size of the obtained nanoparticles were about300 nm, about 400 nm and about 800 to 900 nm, respectively.

The nanoparticle of a carrier encapsulated with CdtB of the presentinvention can effectively inhibit the growth of the radio-resistantprostate cancer cells and increase the sensitivity of theradio-resistant prostate cancer cells to radiation. Without beinglimited by theory, it is believed that CdtB can effectively inhibit thegrowth of prostate cancer cells based on its effect on the DNA of thecancer cells. Because of the high duplication ability and more DNAcontent of the radio-resistant prostate cancer cells, they are easier tobe inhibited by CdtB. Therefore, the use of the nanoparticle of acarrier encapsulated with CdtB in combination with a radiation therapycan provide a better effect on treating prostate cancer.

Accordingly, the nanoparticle of a carrier encapsulated with CdtB of thepresent invention can be used to provide a pharmaceutical compositionfor reducing the radio-resistance of prostate cancer cells which areresistant to radiation or treating prostate cancer, and/or be used inthe manufacture of a medicament for reducing radio-resistance ofprostate cancer cells which are resistant to radiation or treatingprostate cancer, when the treatment is in combination with a radiation.The pharmaceutical composition or the medicament can be manufacturedinto a medicament of any suitable form for administration. Depending onthe form and purpose of the pharmaceutical composition or medicament,the pharmaceutical composition or medicament may further comprise apharmaceutically acceptable carrier.

For example, the pharmaceutical composition or the medicament can bemanufactured into a form suitable for oral administration, subcutaneousinjection, or intravenous injection into a subject. For manufacturing amedicament suitable for oral administration, the pharmaceuticalcomposition or the medicament can comprise a pharmaceutically acceptablecarrier which has no adverse influence on the activity of thenanoparticle of a carrier encapsulated with CdtB, such as a solvent, anoily solvent, a diluent, a stabilizer, an absorption delaying agent, adisintegrant, an emulsifier, an antioxidant, a binder, a lubricant, anda moisture absorbent. The medicament can be prepared as a formulationfor the oral administration by any suitable method, such as a tablet, acapsule, a granule, powder, a fluid extract, a solution, a syrup, asuspension, an emulsion, a tincture, etc.

As for a medicament suitable for subcutaneous injection or intravenousinjection, the medicament or the pharmaceutical composition manufacturedfrom the nanoparticle of a carrier encapsulated with CdtB may compriseone or more components, such as an isotonic solution, a saline buffersolution (e.g. a phosphate buffer solution or a citrate buffersolution), a solubilizer, an emulsifier, other carriers, etc., tomanufacture the medicament as an intravenous injection, an emulsionintravenous injection, a powder injection, a suspension injection, apowder-suspension injection, etc.

In addition to the above adjuvants, the medicament or the pharmaceuticalcomposition manufactured from the nanoparticle of a carrier encapsulatedwith CdtB may comprise other additives, such as a flavoring agent, atoner, a coloring agent, etc. to enhance the taste and visual appeal ofthe resultant medicament or composition. To improve the storability ofthe medicament or the pharmaceutical composition, a suitable amount of apreservative, a conservative, an antiseptic, an anti-fungus reagent,etc. may also be added. Furthermore, the medicament or thepharmaceutical composition may comprise one or more other activecomponents or be used in combination with a medicament comprising theone or more active components to further enhance the efficacy of themedicament or the composition or to increase the application flexibilityand adaptability of the medicament or pharmaceutical composition of thepresent invention, as long as the other active components have noadverse effect on the nanoparticle of a carrier encapsulated with CdtB.

In addition, the medicament or the pharmaceutical compositionmanufactured from the nanoparticle of a carrier encapsulated with CdtBcan be simultaneously or separately administered with a radiationtherapy to the subject in need of such treatment. For example, themedicament or pharmaceutical composition could be administered by oraladministration or injection. The radiation therapy could be performedimmediately or after a time interval (usually after 24-48 hours).Alternatively, radiation therapy could be performed a while before theoral administration or injection of the medicament or the pharmaceuticalcomposition, and be performed again after the oral administration orinjection.

The medicament or the pharmaceutical composition manufactured from thenanoparticle of a carrier encapsulated with CdtB can be administeredwith various administration frequencies, such as once daily, severaltimes a day, or once every few days, etc. For example, when applied tothe human body for reducing the radio-resistance of prostate cancercells, the nanoparticle of a carrier encapsulated with CdtB isadministrated at a dosage ranging from about 1 mg (as CdtB)/kg-bodyweight to about 5 mg (as CdtB)/kg-body weight per day, and preferablyfrom about 2.5 mg (as CdtB)/kg-body weight to about 4 mg (asCdtB)/kg-body weight per day, wherein the unit “mg/kg-body weight” meansthe dosage required per kg-body weight of the treated subject. When themedicament or the pharmaceutical composition manufactured from thecarrier encapsulated with CdtB is administered to a subject once a dayin combination with a radiation therapy to treat prostate cancer, thenanoparticle of a carrier encapsulated with CdtB is administrated at anamount ranging from about 1 mg (as CdtB)/kg-body weight to about 5 mg(as CdtB)/kg-body weight, and preferably about 1.5 mg (as CdtB)/kg-bodyweight to about 3 mg (as CdtB)/kg-body weight. For example, acombination of the medicament or the pharmaceutical composition and theradiation therapy can provide an excellent treatment efficacy as themedicament or pharmaceutical composition is administered at an amount ofabout 2.5 mg (as CdtB)/kg-body weight once a day. However, for patientswith acute conditions, the dosage can be increased to several times orseveral tens of times, depending on the practical requirements.

The present invention also provide a method for reducing theradio-resistance of prostate cancer cells which are resistant toradiation in a subject, comprising administering to the subject aneffective amount of an active component, wherein the active component isthe nanoparticle of a carrier encapsulated with CdtB. The types ofcarrier and the applied form and the dosage of the nanoparticle of acarrier encapsulated with CdtB are all as described hereinabove.

The present invention also provides a method for treating prostatecancer in a subject, comprising administering to the subject aneffective amount of an active component and radiation therapy, whereinthe active component is a nanoparticle of a carrier encapsulated withCdtB. The types of carriers, applied forms and dosages of thenanoparticle of a carrier encapsulated with CdtB are all as describedabove.

The present invention will be further illustrated in details withspecific examples as follows. However, the following examples areprovided only for illustrating the present invention, and the scope ofthe present invention is not limited thereby.

Example 1 Preparation of Nanoparticles of a Carrier Encapsulated withCdtB

First, 0.2 ml CdtB (4.0 mg/ml) and 0.2 ml heparin solution (2.0 mg/ml)were mixed to provide a mixture. The mixture was stirred in 2 mlchitosan solution (1.5 mg/ml) at room temperature for 10 minutes. Then,the mixture was centrifuged at 15,000 g for 50 minutes, and thesupernatant liquid was removed and the precipitates (i.e. the carrierencapsulated with CdtB particles) were collected.

The collected precipitates were dissolved in deionized water to obtain asuspension. The form of the particles in the suspension was observedthrough a transmission electron microscope (TEM) and the distribution ofthe particle size was analyzed by dynamic light scattering spectrometer(Zetasizer ZS90, Malvern, United Kingdom). The results are shown inFIGS. 1A and 2. As shown in FIGS. 1A and 2, the carrier encapsulatedwith CdtB particles were smaller than 500 nm, belonging tonanoparticles, and the average particle size of the carrier encapsulatedwith CdtB particles was about 300 nm (hereinafter referred to as “thenanoparticle of a carrier encapsulated with CdtB”).

In addition, particles containing no CdtB were manufactured by the sameprocedures without using CdtB. The form of the particles in thesuspension was observed through a transmission electron microscope (TEM)and shown in FIG. 1B. As showed in FIG. 1B, the particle size of thenanoparticles of a carrier encapsulated without CdtB was about 200 nm to250 nm.

Example 2 The Cytotoxicity Experiment of the Cancer Cells

To evaluate whether the nanoparticle of a carrier encapsulated with CdtBcould affect the viability of the prostate cancer cells,1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan (MTT) was used in thisexperiment.

MTT is a soluble tetrazolium salt and can affect the respiratory chainof the mitochondria in live cells. Under the effect of succinatedegydrogenase (SDH) and cytochrome c (cyt c), tetrazolium bromide in MTTstructure would be a metabolic reduction and an ianthinus formazon withan insoluble crystal would be formed. Because of the absence of thesuccinate dehydrogenase in dead cells, the MTT can not be reduced, andthe formation of the crystal substrate is also considered to be directlyproportionate to the number of live cells. Furthermore, themitochondrion is an organelle which is the most sensitive to theenvironment, and thus, it can be used as a marker for analyzing cellviability after the medicament treatment.

The prostate cancer cells (5×10³ cells/well) with radio-resistance(PC3-KD cell line with the DAB2IP gene deletion) were cultured in a96-well culture plate. After being cultured in an incubator at 5% CO₂ at37□ for 24 hours, the cells were treated individually with the followingmethod: (i) treated with 500 nM nanoparticles of carrier encapsulatedwithout CdtB for 24 hours (hereinafter referred to as a “controlgroup”); (ii) treated with 500 nM nanoparticle of a carrier encapsulatedwith CdtB for 24 hours (hereinafter referred to as “CdtB nanoparticlegroup”); (iii) irradiated with 2 gray (Gy) radiation for 3 minutes(hereinafter referred to as “radiation group”); and (iv) treated with500 nM nanoparticle of a carrier encapsulated with CdtB, and irradiatedwith 2 Gy radiation for 3 minutes and reacted for 24 hours (hereinafterreferred to as “CdtB nanoparticle+radiation group”). After the abovetreatment, the suspension was removed. Then, 10 μl/well of MTT and 100μl/well of culture medium were added in the dark for 4 hours. Then, theall culture medium was removed; added 100 μl/well of DMSO in the dark toreact for 10 minutes and analyzed the wavelength of 570 nm absorbance.The absorbance of the “control group” (viability is 100%) was used as acontrol, and the viability of each group were measured. The results areshown in FIG. 3 and Table 1.

TABLE 1 Group Cancer cell viability (%) control group 100 CdtBnanoparticle group 69.73 radiation group 95.30 CdtB nanoparticle +radiation 55.4

According to FIG. 3 and Table 1, the viability of cancer cells in“radiation group” was still up to 95.30%, revealing that the prostatecancer cells had radio-resistance could not be effectively killed byradiation treatment alone. The result of the “CdtB nanoparticle group”showed that the viability of the prostate cancer cells withradio-resistance was reduced to 69.73% when treated with the CdtBnanoparticle alone, indicating that the CdtB nanoparticle was effectivein treating prostate cancer cells. The result of the “CdtBnanoparticle+radiation group” showed the viability of prostate cancercells with radio-resistance was reduced to 55.4% when treated with “CdtBnanoparticle” in combination with “radiation,” indicating that the “CdtBnanoparticle” had the ability to reduce the radio-resistance of prostatecancer cells, and thus, can reduce the viability of cancer cells from69.73% to 55.4% when treated with “CdtB nanoparticle” in combinationwith “radiation” rather than treated with “CdtB nanoparticle” alone.

What is claimed is:
 1. A method for reducing the radio-resistance ofprostate cancer cells in a subject in need thereof, comprisingadministering to the subject an effective amount of an active component,wherein the active component is a nanoparticle of a carrier encapsulatedwith cytolethal distending toxin subunit B (CdtB), and the carriercomprises chitosan and heparin.
 2. The method according to claim 1,wherein the nanoparticle has a particle size not greater than 1000nanometers.
 3. The method according to claim 2, wherein the nanoparticlehas a particle size not greater than 500 nanometers.
 4. The methodaccording to claim 1, wherein the nanoparticle has an average particlesize of about 300 nanometers to about 400 nanometers.
 5. The methodaccording to claim 1, wherein the cytolethal distending toxin subunit Bprotein is cloned from Campylobacter jejuni.
 6. A method for treatingprostate cancer in a subject in need thereof, comprising simultaneouslyor separately administering to the subject a radiation therapy and aneffective amount of an active component, wherein the active component isa nanoparticle of a carrier encapsulated with cytolethal distendingtoxin subunit B (CdtB), and the carrier comprises chitosan and heparin.7. The method according to claim 6, wherein the nanoparticle has aparticle size not greater than 1000 nanometers.
 8. The method accordingto claim 7, wherein the nanoparticle has a particle size not greaterthan 500 nanometers.
 9. The method according to claim 6, wherein thenanoparticle has an average particle size of about 300 nanometers toabout 400 nanometers.
 10. The method according to claim 6, wherein thecytolethal distending toxin subunit B protein is cloned fromCampylobacter jejuni.